The Pathogenic Diagnosis in Pediatric Diabetology: Next Generation Sequencing and Precision Therapy

Abstract In pediatric diabetology, a precise diagnosis is very important because it allows early and correct clinical management of the patient. Monogenic diabetes (MD), which accounts for 1–6% of all pediatric–adolescent diabetes cases, is the most relevant example of precision medicine. The definitive diagnosis of MD, possible only by genetic testing, allows us to direct patients to more appropriate therapy in relation to the identified mutation. In some cases, MD patients can avoid insulin and be treated with oral hypoglycemic drugs with a perceptible impact on both the quality of life and the healthcare costs. However, the genetic and phenotypic heterogeneity of MD and the overlapping clinical characteristics between different forms, can complicate the diagnostic process. In recent years, the development of Next-Generation Sequencing (NGS) methodology, which allows the simultaneous analysis of multiple genes, has revolutionized molecular diagnostics, becoming the cornerstone of MD precision diagnosis. We report two cases of patients with clinical suspects of MD in which a genetic test was carried out, using a NGS multigenic panel, and it clarified the correct pathogenesis of diabetes, allowing us to better manage the disease both in probands and other affected family members

Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a newly discovered second messenger that gates two pore channels 1 (TPC1) and 2 (TPC2) to elicit endo-lysosomal (EL) Ca2+ release. NAADP-induced lysosomal Ca2+ release may be amplified by the endoplasmic reticulum (ER) through the Ca2+-induced Ca2+ release (CICR) mechanism. NAADP-induced intracellular Ca2+ signals were shown to modulate a growing number of functions in the cardiovascular system, but their occurrence and role in cardiac mesenchymal stromal cells (C-MSCs) is still unknown. Herein, we found that exogenous delivery of NAADP-AM induced a robust Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store with Gly-Phe β-naphthylamide, nigericin, and bafilomycin A1, and blocking TPC1 and TPC2, that are both expressed at protein level in C-MSCs. Furthermore, NAADP-induced EL Ca2+ release resulted in the Ca2+-dependent recruitment of ER-embedded InsP3Rs and SOCE activation. Transmission electron microscopy revealed clearly visible membrane contact sites between lysosome and ER membranes, which are predicted to provide the sub-cellular framework for lysosomal Ca2+ to recruit ER-embedded InsP3Rs through CICR. NAADP-induced EL Ca2+ mobilization via EL TPC was found to trigger the intracellular Ca2+ signals whereby Fetal Bovine Serum (FBS) induces C-MSC proliferation. Furthermore, NAADP-evoked Ca2+ release was required to mediate FBS-induced extracellular signal-regulated kinase (ERK), but not Akt, phosphorylation in C-MSCs. These finding support the notion that NAADP-induced TPC activation could be targeted to boost proliferation in C-MSCs and pave the way for future studies assessing whether aberrant NAADP signaling in C-MSCs could be involved in cardiac disorders

Screening and isolation of microbes from a Mud Community of Ischia Island Thermal Springs: preliminary analysis of a bioactive compound

Introduction: Balneotherapy centers of Ischia island (Italy) offer treatments for different dermatological diseases (psoriasis, acne, atopic dermatitis) and upper respiratory tract infections. In this study, we integrated morphological and molecular approaches to give bacterial diversity of microbial mat samples collected from the thermae of Ischia. Methods: Samples were collected during 2019 at four sites. Some bacterial strains ATCC for antibacterial and antibiofilm Activity were tested. After morphological characterization, screening for antagonistic isolates was made. The colonies isolated from thermal mud samples were submitted to molecular characterization. Susceptibility testing by dilution spotting was carried out and antibacterial efficacies of most active isolate were evaluated with a Minimal inhibition concentration assay. Biofilm formation, inhibition, eradication were examined. Statistical analyses were carried out utilizing Microsoft® Excel 2016/XLSTAT©-Pro. Results: We isolated a natural compound with antimicrobial and antibiofilm activities. Conclusions: The results obtained in this study are discussed in the context of how hydrothermal systems are important environmental source of uncharted antimicrobial and antibiofilm compounds. This is, to our knowledge, the first view of a spring water microbiome analysis of Ischia

Candida Biofilm Eye Infection: Main Aspects and Advance in Novel Agents as Potential Source of Treatment

Abstract: Fungi represent a very important cause of microbial eye infections, especially in tropical and developing countries, as they could cause sight-threating disease, such as keratitis and ocular candidiasis, resulting in irreversible vision loss. Candida species are among the most frequent microorganisms associated with fungal infection. Although Candida albicans is still the most frequently detected organism among Candida subspecies, an important increase in non-albicans species has been reported. Mycotic infections often represent an important diagnostic-clinical problem due to the difficulties in performing the diagnosis and a therapeutic problem due to the limited availability of commercial drugs and the difficult penetration of antifungals into ocular tissues. The ability to form biofilms is another feature that makes Candida a dangerous pathogen. In this review, a summary of the state-of-the-art panorama about candida ocular pathology, diagnosis, and treatment has been conducted. Moreover, we also focused on new prospective natural compounds, including nanoparticles, micelles, and nanocarriers, as promising drug delivery systems to better cure ocular fungal and biofilm-related infections. The effect of the drug combination has also been examined from the perspective of increasing efficacy and improving the course of infections caused by Candida which are difficult to fight

Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation

The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC–CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal‐like hiPSC‐CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC‐CMs maturity, spanning from long‐term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.Fil: Lodola, Francesco. Università degli Studi di Milano; ItaliaFil: de Giusti, Verónica Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Maniezzi, Claudia. Università degli Studi di Milano; ItaliaFil: Martone, Daniele. Università degli Studi di Milano; ItaliaFil: Stadiotti, Ilaria. Università degli Studi di Milano; ItaliaFil: Sommariva, Elena. Università degli Studi di Milano; ItaliaFil: Maione, Angela Serena. Università degli Studi di Milano; Itali

Synthesis of temporin L hydroxamate-based peptides and evaluation of their coordination properties with iron (III)

Ferric iron is an essential nutrient for bacterial growth. Pathogenic bacteria synthesize iron-chelating entities known as siderophores to sequestrate ferric iron from host organisms in order to colonize and replicate. The development of antimicrobial peptides (AMPs) conjugated to iron chelators represents a promising strategy for reducing iron availability, inducing bacterial death, and enhancing simultaneously the efficacy of AMPs. Here we designed, synthesized, and characterized three hydroxamate-based peptides Pep-cyc1, Pep-cyc2, and Pep-cyc3, derived from a cyclic temporin L peptide (Pep-cyc) developed previously by some of us. The Fe3+ complex formation of each ligand was characterized by UVvisible spectroscopy, mass spectrometry, IR, and NMR spectroscopies. In addition, the effect of Fe3+ on the stabilization of -helix conformation of hydroxamate-based peptides and the cotton effect were examined by CD spectroscopy. Moreover, the antimicrobial results obtained in vitro on some Gram-negative strains (K. Pneumoniae and E. coli) showed the ability of each peptide to chelate efficaciously Fe3+ obtaining a reduction of MIC values in comparison to their parent peptide Pepcyc. Our results demonstrated that siderophore conjugation could increase the efficacy and selectivity of AMPs used for the treatment of infectious diseases caused by Gram-negative pathogens

Allium ursinum and Allium oschaninii against Klebsiella pneumoniae and Candida albicans Mono- and Polymicrobic Biofilms in In Vitro Static and Dynamic Models

The present study assesses the in vitro antibiofilm potential activity of extracts of wild Allium ursinum and Allium oschaninii. The active ingredients of the extracts were obtained with a technique named Naviglio (rapid solid–liquid dynamic extraction, RSLDE) which is based on an innovative and green solid–liquid extraction methodology. The extracts were tested against models of mono‐ and polymicrobial biofilm structures of clinically antibiotic‐resistant pathogens, Klebsiella pneumoniae ATCC 10031 and Candida albicans ATCC 90028. Biofilms were studied using a static and a dynamic model (microtiter plates and a CDC reactor) on three different surfaces reproducing what happens on implantable medical devices. Antimicrobic activities were determined through minimum inhibitory concentration (MIC), while antibiofilm activity was assessed by minimum biofilm eradication concentration (MBEC) using a crystal violet (CV) biofilm assay and colony forming unit (CFU) counts. Results showed that both Allium extracts eradicated biofilms of the tested microorganisms well; biofilms on Teflon were more susceptible to extracts than those on polypropylene and polycarbonate, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. Our data provide significant advances on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications

Activity of Free and Liposome-Encapsulated Essential Oil from Lavandula angustifolia against Persister-Derived Biofilm of Candida auris

The high virulence of Candida auris, a pathogen fungus considered as a global threat for public health, is due to its peculiar traits such as its intrinsic resistance to conventional antifungals. Its biofilm lifestyle certainly promotes the prolonged survival of C. auris after disinfection or antifungal treatments. In this work, for the first time, we detected persister cells in a biofilm of C. auris in a microwell plate model, following caspofungin treatment. Furthermore, we showed how persisters can progressively develop a new biofilm in situ, mimicking the re-colonization of a surface which may be responsible for recalcitrant infections. Plant-derived compounds, such as essential oils, may represent a valid alternative to combat fungal infections. Here, Lavandula angustifolia essential oil, as free or encapsulated in liposomes, was used to eradicate primary and persister-derived biofilms of C. auris, confirming the great potential of alternative compounds against emergent fungal pathogens. As in other Candida species, the action of essential oils against C. auris involves ROS production and affects the expression of some biofilm-related genes

Antifungal and Antibiofilm Activity of Cyclic Temporin L Peptide Analogues against Albicans and Non-Albicans Candida Species

Temporins are one of the largest families of antimicrobial peptides with both anti-inflammatory and antimicrobial activity. Herein, for a panel of cyclic temporin L isoform analogues, the antifungal and antibiofilm activities were determined against representative Candida strains, including C. albicans, C. glabrata, C. auris, C. parapsilosis and C. tropicalis. The outcomes indicated a significant anti-candida activity against planktonic and biofilm growth for four peptides (3, 7, 15 and 16). The absence of toxicity up to high concentrations and survival after infection were assessed in vivo by using Galleria mellonella larvae, and the correlation between conformation and cytotoxicity was investigated by fluorescence assays and circular dichroism (CD). By combining fluorescence spectroscopy, CD, dynamic light scattering, confocal and atomic force microscopy, the mode of action of four analogues was hypothesized. The results pinpointed that peptide 3 emerged as a non-toxic compound showing a potent antibiofilm activity and represents a promising compound for biomedical applications